The present invention relates to a method for measuring microparticles and an instrument for measuring microparticles capable of detecting microparticles having a very low fluorescence intensity existing in liquid with a high precision to analyze them as well as to a quantitative measuring method and an instrument for realizing the same, useful in fields of immunological analysis and genetic diagnosis using microparticles.
(a) As a method for analyzing particles, etc., there is known an analyzing method using a flow cytometer. In the flow cytometer, liquid containing particles is led to a sheath flow cell so that particles flow separately one after another through an approximately central portion thereof, the particles flowing therethrough are irradiated with laser light, and scattered light or fluorescence (in the case where the particles are fluorescent) thus produced is detected by means of a photodetector such as a photomultiplier, etc. By this method the property of the particles can be analyzed by using pulse heights or integrated values of an output signal of the photodetector as a function of a time for the passage of a particle through the laser light. In order to increase the analysis precision by such a method, there are known a method, by which pulse width information is added to the pulse height, as described e.g. in JP-A-Sho-58-41336, and a method, by which the time of starting analysis of the pulse height or the integrated value is substantially advanced by delaying the signal pulses, as described in JP-A-SHo-65-35345.
(b) On the other hand, as an immunological measurement method using particles, there is known a method, by which antigen concentration is measured by making latex spheres, with the surface of which an antibody is bound, react with an antigen and measuring the agglutinated state of the latex spheres produced by the antigen-antibody reaction by the absorbance or the intensity of scattered light. Further, in order to analyze this agglutinated state with a high precision, there is known also a method, by which each agglutinated lump is led to a flow cytometer to be analyzed there. By this method it is possible to calculate the magnitude of each agglutinated lump, based on the intensity of scattered light to measure the antigen concentration with a high precision. This method is described e.g. in "Kensa to Gijutsu (Test and Techniques)" Vol. 16, No. 7 (1988), pp. 607-613.
(c) Further, as described e.g. in JP-A-SHo-56-151357, there is proposed a method by which particles having a diameter of about 0.5 .mu.m are made to bind with an antigen to be measured by a reaction of a heterogenous system to measure the antigen concentration while counting the number of particles by means of a microscope.